The Isolation, Preservation, and Improvement of Industrial

Micro-organisms

Jumeri M. Wikarta, Ph.D

The Isolation of IndustriallyImportant Micro-organisms

Isolation : - Obtaining of either pure/mixed cultures - Assessment to determine which

carry out the desired reaction or produce the desired product

- The isolate must carry out the process economically

- The selection of the culture to be used is a compromise between the productivity of the organism and the economic constraints of the process

Bull et al. (1979) : criteria as being important in the choice of organism :

The nutritional characteristics of the organism → very cheap medium or predetermined

The optimum temperature of the organism → above 40°C

The reaction of the organism with the equipment to be employed and the suitability of the organism to the type of process to be used

Bull et al. (1979) : criteria as being important in the choice of organism :

The stability of organism and its amenability to genetic manipulation

The productivity of the organism, measured in its ability to convert substrate into product and to give a high yield of product per unit time

The ease of product recovery from the culture

Assesment the product and organism

toxicity

Isolation of cultures Natural environments - myriad of organisms, very few of which may be satisfactory - superior organism may be found Culture collection - known characteristic - may not contain those possessing

the most desirable features - cheaper

Major Culture Collection

National Collection of Type Cultures (NCTC) – London

National Collections of Industrial and Marine Bacteria (NCIMB) – UK

American Type Culture Collection (ATCC) – Maryland

Deutsche Sammlung von Micro-organismen (DSM) – Gottingen

Isolation method utilizing selection of the desired characteristic

Enrichment Liquid Culture→ A technique resulting in an increase in the number of a given microorganism relative to the numbers of other types in the original inoculum→ taking a mixed population→ Providing condition (particular

substrate, certain inhibitor)

The prevalence of an organism in an enrichment culture will depend on its maximum specific growth rate

→ Enrichment broth is sub-cultured at the correct time→ The dominant organism will be the fastest growing

Isolation method utilizing selection of the desired characteristic

It is not necessarily true that the organism with the highest specific growth rate is the most useful

May be desirable to isolate the organism with the highest affinity for its substrate

The problem of time of transfer and selection on the basis of maximum specific growth rate

Isolation method utilizing selection of the desired characteristic

Overcome : By the use of a continuous process Fresh medium is added to the culture at

a constant rate Dominan organism will be selected on

the basis of its afinity for the limiting substrate

Growth rate is controlled by the dilution rate and is related to the limiting substrate concentration μmax

s

μ = Ks + S

Residual substrate concentration

μmax

Ks

1/2μmax

Effect of substrate conc. on the specific growth rate of micro-organism

In continuous culture, the specific growth rate is determined by the substrate conc. and is equal to the dilution rate

If A and B were present in a continuous enrichment culture, limited by the substrate, Strain A would be selected at dilution rate obove YStrain B would be selected at dilution rate below Y

The organism which are isolated by continuous enrichment culture will depend on the dilution rate

Continuous enrichment techniques are especially valuable in isolating organisms to be used in a continuous-flow commercial process

Harrison (1976) : Organism isolated by batch enrichment and purification on solid media frequently perform poorly in continuous culture

The enrichment procedure should be designed such that the predicted isolates meet as many of the criteria of the propose process as possible

Johnson (1972) and Harrison (1975)

Procedure for the isolation of organism to be used for biomass production

Johnson : using the carbon source, with vitamin or yeast extract

Consideration in the design of a commercial continuous process :

- simple medium - cheaper commercial process - resistant to contamination - the use of as high as possible an isolation temperature

Main difficulty in Continuous-enrichment process : Washout of the inoculum before an adapted culture is established

Johnson (1972) :Isolation process started in batch culture using 20 % inoculum

As soon as growth is observed

The culture transferred to fresh medium

Subsequent purification and stabilization in continuous culture

Harrison (1978) :

Use a turbidostat :

Continuous-flow system with a photoelectric cell to determine the turbidity of the culture and maintain turbidity between set point by initialing or terminating the addition of medium

Wash out is avoided as the medium supply will be swicth off if the biomass falls below the lower fixed point

Turbidostat : - operates at high levels of limiting substrate - removes the danger of wash out - It is not as flexible a system as the chemostat (used at a range of dilution rate)

Use a two-stage chemostat bjg

The first stage was used as a continuous inoculum for the second stage and consisted of a large bottle containing a basic medium inoculated with a soil infusion

Continuous inoculation was employed until an increasing absorbance was observed in the second stage

Continuous enrichment

culture

Resulted in the selection of stable, mixed culture presumably based on some form of symbiotic relationship

May result in the development of novel, mixed culture fermentation

Also used for the isolation of organisms to be used in system other than biomass production

Rolley and Bull (1977) : isolate an Arthrobacter sp producing a yeast lysing enzyme complex